Use of polypeptides obtained through systematic mutations of single amino acids of human and non-human Box-A of HMGB1 to prevent and/or antagonize pathologies induced by HMGB1

ABSTRACT

The present invention relates to polypeptide variants of the HMGB-1 high affinity binding domain Box-A (HMGB1 Box-A) or to a biologically active fragment of HMGB1 Box-A, which are obtained through systematic mutations of single amino acids of the wild-type HMGB1 Box-A protein and which show an increased resistance to proteases and which are therefore characterized by more favourable pharmacokinetic and pharmacodynamic profiles. Moreover, the present invention concerns the use of said polypeptide molecules of HMGB1 Box-A to diagnose, prevent, alleviate and/or treat pathologies associated with extracellular HMGB1 and associated with RAGE.

The present invention relates to polypeptide variants of the HMGB-1 highaffinity binding domain Box-A (HMGB1 Box-A) or to a biologically activefragment of HMGB1 Box-A, which are obtained through systematic mutationsof single amino acids of the wild-type HMGB1 Box-A protein and whichshow an increased resistance to proteases and which are thereforecharacterized by more favourable pharmacokinetic and pharmacodynamicprofiles. Moreover, the present invention concerns the use of saidpolypeptide molecules of HMGB1 Box-A to diagnose, prevent, alleviateand/or treat pathologies associated with extracellular HMGB1 andassociated with the receptor RAGE.

Recent research in the field of sepsis and inflammation has led to animproved understanding of the pathogenic mechanisms and eventsunderlying their clinical onset and development. In the early stages ofsepsis, for instance, bacterial endotoxins stimulate cells of the innateimmune system which release pro-inflammatory cytokines (TNF, IL-1α andIL-6). These early cytokines in turn induce the release of alater-acting downstream mediator (identified as the known protein HMGB1)that triggers the pathological sequelae mediated by the subsequentrelease of cytokines such as TNF, IL-1α, IL-1β, IL-1Ra, IL-6, IL-8,IL-18, IFN-γ, PAF, etc., leading to a multisystem pathogenesis or to alethal systemic inflammation (Andersson et al., 2002).

The HMGB1 protein belongs to the family of high mobility group (HMG)proteins. HMG proteins, so-called due to their high electrophoreticmobility in polyacrylamide gels, are the most ubiquitous non-histoneproteins associated with isolated chromatin in eukaryotic cells. Theseproteins play a generalized “architectural” role in DNA bending,looping, folding and wrapping, since they either distort, bend or modifyDNA structures and complexes with transcription factors or histones(Andersson et al., 2002; Agresti et al., 2003; Degryse et al., 2003).The high mobility group 1 (HMGB1) protein is usually a nuclear factor,in particular a transcriptional regulatory molecule causing DNA bendingand facilitating the binding of several transcriptional complexes.

Structurally, the HMGB1 protein is a protein of approximately 25 kDawith a highly conserved sequence among mammals, whereby 2 out of 214amino acids have conservative substitutions in all mammalian species.HMGB1 is ubiquitously present in all vertebrate nuclei and in particularcan be found in fibroblasts, neurons, hepatocytes, glia and in cellsderived from hematopoietic stem cells, including monocytes/macrophages,neutrophils and platelets. The HMGB1 molecule has a tripartite structurecomposed of three distinct domains: two DNA binding domains called HMGBox-A and Box-B, and an acid carboxyl terminus, making it bipolarlycharged.

The two HMGB1 boxes are involved in the protein's function asnon-sequence-specific architectural DNA-binding elements, conferring theability to bind DNA into recognized distorted DNA structures andstabilizing nucleosome assembly, remodelling and sliding. Both the A-and B-HMG boxes are made up of highly conserved 84 amino acid residues,are strongly positively charged and are arranged in three α-heliceshaving a similar L-shaped fold. The long arm of the “L” contains theN-terminal extended strand and helix III (Andersson et al. 2002; Agrestiet al., 2003; Thomas, J. O. 2001), while the short arm comprises helicesI and II. Structure-function analysis reveals that the pro-inflammatorycytokine domain of HMGB1 is the B-Box and in particular the sequence ofits first 20 amino acids. The A-Box is an extremely weak agonist of theinflammatory cytokine release triggered by HMGB1 and competitivelyinhibits the pro-inflammatory activities of the B-Box and of the wholeprotein. Therefore, from a pharmacological point of view, the A-Box actsas an antagonist of the pathological conditions induced and/or sustainedby the B-Box and HMGB1.

The third domain, the carboxyl terminus or acidic tail, is extremelynegatively charged since it contains 30 repetitive aspartic and glutamicacid residues, and is linked to the boxes by a basic region of about 20residues. Mouse and rat HMGB1 differ from the human form by only twosubstitutions that are located in this continuous C-terminal stretch.

HMGB1 binds rather weakly to the B-form variety of lineardouble-stranded DNA with no sequence specificity, while it binds in theinterior of the nucleus with high affinity to supercoiled DNA, tounusual DNA structures like 4-way junctions (cruciform DNA), bulged DNAand bent DNA (Ferrari et al., 1992; Pontiggia et al., 1993 andPCT/EP2005/007198 in the name of Creabilis Therapeutics).

Besides its nuclear location and role as a transcription factorregulator, HMGB1 has also been found in the extracellular medium,actively released by activated cells of the immune systems (monocytesand macrophages) or passively released by damaged or necrotic cells(Andersson et al., 2002; Scaffidi et al., 2002; Bonaldi et al., 2002;Taniguchi et al., 2003; Friedman et al., 2003; Palumbo et al., 2004).

Extracellularly released HMGB1 acts as a potent cytokine and as anextremely potent macrophage-stimulating factor. HMGB1 acts directly bybinding to the cell membrane, inducing signaling and chemotaxis, havinga chemokine-like function (Yang et al., 2001) and further actingindirectly by up-regulating the expression and secretion ofpro-inflammatory cytokines. This makes extracellular HMGB1 protein apotent chemotactic and immunoregulatory protein which promotes aneffective inflammatory immune response. Furthermore, other proteinsbelonging to the family of HMG proteins, and which are able to bend DNA,are released together with HMGB1 in the extracellular medium. Theseproteins are inter alia HMGB2, HMGB3, HMG-1L10, HMG-4L and SP100-HMG.They share with HMGB1 highly homologous amino acid sequences. LikeHMGB1, they trigger/sustain inflammatory pathologies interacting withthe same receptors, leading to the same downstream pathways ofinteraction.

In healthy cells, HMGB1 migrates to the cytoplasm both by passive andactive transport. However, all cultured cells and resting monocytescontain the vast majority of HMGB1 in the nucleus, indicating that inbaseline conditions import is much more effective than export. Cellsmight transport HMGB1 from the nucleus by acetylating lysine residueswhich are abundant in HMGB1, thereby neutralizing their basic charge andrendering them unable to function as nuclear localization signals.Nuclear HMGB1 hyperacetylation determines the relocation of this proteinfrom the nucleus to the cytoplasm (in the fibroblasts, for example) orits accumulation into secretory endolysosomes (in activated monocytesand macrophages, for example) and subsequent redirection towards releasethrough a non-classical vesicle-mediated secretory pathway. HMGB1secretion by already activated monocytes is then triggered by bioactivelysophosphatidylcholine (LPC), which is generated later in theinflammation site from phosphatidylcholine through the action of thesecretory phospholipase sPLA2 produced by monocytes several hours afteractivation. Therefore, secretion of HMGB1 seems to be induced by twosignals (Bonaldi et al., 2003) and to take place in three steps: 1) atfirst, an inflammatory signal promotes HMGB1 acetylation and itsrelocation from the nucleus to the cytoplasm (step 1) and storage incytoplasmic secretory vesicles (step 2); then, a secretion signal(extracellular ATP or lysophosphatidylcholine) promotes exocytosis(third step) (Andersson et al., 2002; Scaffidi et al. 2002; Gardella etal., 2002; Bonaldi et al., 2003; Friedman et al., 2003).

Released HMGB1 has been identified as one of the ligands binding to theRAGE receptor. This receptor is expressed in most cell types, and at ahigh level mainly in endothelial cells, in vascular smooth muscle cells,in monocytes and macrophages and in mononuclear phagocytes. Recognitioninvolves the C-terminal of HMGB1. The interaction of HMGB1 and RAGEtriggers a sustained period of cellular activation mediated by RAGEup-regulation and receptor-dependent signaling. In particular, theinteraction of HMGB1 and RAGE activates several intracellular signaltransduction pathways, including mitogen-activated protein kinases(MAPKs), Cdc-42, p21ras, Rac and the nuclear translocation factor κB(NF-κB), the transcription factor classically linked to inflammatoryprocesses (Schmidt et al., 2001).

According to several experimental evidences, released HMGB1 may alsointeract with receptors belonging to one or more subclasse(s) of thefamily of the Toll-like receptors. Further, HMGB1 may also interact withthe functional N-terminal lectin-like domain (D1) of thrombomodulin. Dueto the ability of the functional D1 domain of thrombomodulin tointercept and bind circulating HMGB1, the interaction with the RAGEreceptors and the Toll-like receptors is prevented.

In the context of the present invention, “HMGB1” includes thenon-acetylated form or/and the acetylated form of HMGB1. Likewise,“HMGB1 homologous proteins” include the non-acetylated form or/and theacetylated form of HMGB1 homologous proteins. Preferred HMGB1 homologousproteins are HMGB2, HMGB3, HMG-1L10, HMG-4L or/and SP100-HMG.

When released in vivo, HMGB1 is an extremely potent cytokine and apotent macrophage-stimulating factor. In fact, like other cytokinemediators of endotoxemia, HMGB1 activates in vitro a cascade of multiplepro-inflammatory cytokines (TNF, IL-1α, IL-1β, IL-1Ra, IL-6, IL-8,MIP-1α and MIP-1β) from human macrophages. Therefore, HMGB1 acts as alate mediator during acute inflammation and participates in an importantway in the pathogenesis of systemic inflammation after the earlymediator response has been resolved.

The observed pro-inflammatory effects of HMGB1 in vitro and thecorrelation between circulating HMGB1 levels and the development of thepathogenic sequence of systemic inflammation in vivo indicate thattherapeutically targeting of this cytokine-like molecule should be ofrelevant clinical value, suggesting novel therapeutic approaches by a“late” administration of (selective) antagonists/inhibitors of theextracellular activities of HMGB1.

Moreover the observed RAGE upregulation in proinflammatory environmentsand the proved increased expression of this receptor in a variety ofacute and chronic inflammatory diseases provide support for RAGE as anattractive target for future clinical interventions related toinflammation.

Therefore, several attempts were performed in order to block thisextracellular HMGB1 chemo-cytokine protein. Several important approacheswere addressed to the administration of antibodies against HMGB1, ofHMGB1 fragments (for example HMGB1 A-Box), of antibodies to RAGE, ofsoluble RAGE (sRAGE) and of ethyl pyruvate (Czura et al., 2003; Lotze etal., 2003).

The passive immunization of mice with HMGB1-neutralizing antibodiesconferred a highly significant, dose-dependent and lasting protectionagainst lethal doses of endotoxin, even when the first doses ofantibodies were given after the TNF peak had passed, suggesting thatantagonizing HMGB1 activity late in the clinical course may be aneffective treatment approach to potentially lethal sepsis (Yang et al.,2004).

Another possibility is to administer mono- or oligoclonal antibodiesagainst the HMGB1 B-Box, or its 20 amino acid relevant core whichsignals through RAGE. Furthermore, HMGB1 A-Box, one of the twoDNA-binding domains in HMGB1, has been identified as a specificantagonist of HMGB1: highly purified recombinant A-Box has protectedmice from lethal experimental sepsis even when initial treatment hasbeen delayed for 24 hours after pathology induction, further suggestingthat HMGB1 antagonists may be administered successfully in a clinicallyrelevant window wider than the one used for other known cytokines (Yanget al., 2004).

Structural function analysis of HMGB1-truncated mutants has revealedthat the A-Box domain of HMGB1 competitively displaces the saturablebinding of HMGB1 to macrophages, specifically antagonizing HMGB1activities. As has been already seen with the protective activity ofanti-HMGB1 antibodies, the administration of the A-Box rescues mice fromsepsis even when treatment has been initiated as late as 24 hours aftersurgical induction of sepsis (Yang H. et al., 2004). HMGB1 antagonistsor inhibitors selected from the group of antibodies or antibodyfragments that bind to an HMGB1 protein, HMGB1 gene antisense sequencesand HMGB1 receptor antagonists are known from U.S. Pat. No. 6,468,533,WO 02/074337 and US 2003/0144201.

Moreover, saturation of circulating HMGB1 by the administration of sRAGEleads to the block of its activities mediated by cellular RAGE, a resultwhich can also be obtained by inhibiting RAGE itself with theadministration of anti-RAGE antibodies.

Furthermore, a similar protective response late in the course of sepsishas been observed by administering ethyl-pyruvate, a stable lipophilicderivative and relatively non-toxic food additive also used as anexperimental anti-inflammatory agent, which attenuates the systemicinflammation of ischemia/reperfusion tissue injury and lethalhemorrhagic shock. Ethyl-pyruvate inhibited HMGB1 and TNF release invitro from endotoxin-stimulated murine macrophages, while in vivoprotected mice from peritonitis-induced lethal sepsis, again when dosingwas begun 24 hours after this pathology was experimentally induced.

Finally, it has been shown that the N-terminal lectin-like domain (D1)of thrombomodulin is an inhibitor of HMGB1, since it binds to andsequesters this chemokine, preventing the binding of HMGB1 to RAGE andToll-like receptors such that the downstream cascade of events leadingto inflammatory pathologies is inhibited.

As described above, several attempts were performed with the aim ofinhibiting and/or antagonising the extracellular HMGB1 chemo-cytokineprotein. The present invention is based on the experimental evidencethat the two high affinity binding domains for DNA, i.e. HMGB1 Box-A andHMGB1 Box-A, which are present in the HMGB1 molecule, have two opposingroles in the protein released in the extracellular space. The mainactivity of HMGB1 Box-B is to mediate the pro-inflammatory activitiesattributed to the HMGB1 protein. On the other hand, HMGB1 Box-A acts asan antagonist competing with the pro-inflammatory activity of the Box-Bdomain.

The problem underlying the present invention was therefore the provisionof novel agents for the prevention, alleviation and/or treatment ofHMGB1-associated pathologies. In particular, the problem of the presentinvention was to develop novel agents as selective extracellular HMGB1antagonist and/or inhibitors, in order to prevent, alleviate and/ortreat the broad spectrum of pathological effects induced by the HMGB1chemokine itself and/or by the cascade of multiple inflammatorycytokines caused by the extracellular release of the HMGB1 protein.

The inventors of the present invention based their studies on theevidence that HMGB1 Box-A behaves as an antagonist in the pathologicalconditions induced and/or sustained by HMGB1 and as inhibitors of RAGE.Hence HMGB1 Box-A represents a high potential therapeutic approach toblock both the contribution of HMGB1 and of RAGE to inflammatorydisorders.

However, the delivery of therapeutic proteins for clinical use is amajor challenge to pharmaceutical science. Most natural proteins, assuch, are inefficient drugs due to sub-optimal performance in terms ofpotency, stability, immunogenicity, side effects or toxicity. Once inthe blood stream, these proteins are constantly eliminated fromcirculation within a short time. Different physiological processesinvolving metabolism as well as clearance through normal pathways ofprotein elimination (such as glomerular filtration in the kidneys orproteolysis in blood) are involved. Consequently, also the therapeuticuse is limited by a very short half-life in plasma.

Hence the need to identify a HMGB1 Box-A variant specifically designedto maintain the same or a higher pharmacological activity of wild typebut characterized by a consistent increased resistance to proteases andconsequently improved pharmacokinetic and pharmacodynamic performanceexists in the art.

The solution to the above problem is therefore the provision of apolypeptide variant of the human and/or non-human HMGB1 high affinitybinding domain Box-A (HMGB1 Box-A) or of a biologically active fragmentof human and/or non-human HMGB1 Box-A, characterized in that the aminoacid sequence of said polypeptide variant differs from the amino acidsequence of the wild type HMGB1 Box-A protein by the mutation of one ormore single amino acids. Surprisingly, it was found by the inventors ofthe present invention that said polypeptide variant exhibits anincreased resistance to proteolysis compared to wild type HMGB1 Box-A orto the biological active fragment of the wild type HMGB1 Box-A togetherwith the maintenance of the same or development of even higherpharmacological activity.

By increasing the resistance to the proteolytic activity of theproteases, a more favourable pharmacokinetic and pharmacodynamic profilecan be achieved, since an increased stability in body fluids is obtainedfor the inventive polypeptide variants. As a result thereof, an increasein the half-life in body fluids of the protein's variants of the presentinvention is observed as well. It is known that the estimated half-lifeof proteins in vivo can be as short as a few minutes. The variants ofthe present invention preferably have an increased half-life, e.g.because they are more resistant to proteases.

In a most preferred embodiment of the present invention, polypeptidevariants are obtained by using a directed evolution process, whichtechnology is extensively described in WO 2004/7022593 and in severalfurther patent applications (PCT/FR00/03503, PCT/FR01/01366, U.S. Ser.No. 10/022,249, US10/022,390, U.S. Ser. No. 10/375,192, U.S. 60/409,898,U.S. 60/457,135, U.S. 60/410,258 and U.S. 60/410,263), all in the nameof Nautilus Biotech S. A. (Paris, France), which are herein incorporatedby reference.

In general, the term “directed evolution” refers to biotechnologicalprocesses devoted to the improvement of target protein features by meansof specific changes introduced into their amino acid sequences. Thedirected evolution process includes the generation of a library ofmutant versions of the gene of interest, followed by the selection ofthose variants that display the desired features. These processes can beiterative when gene products having an improvement in a desired propertyare subjected to further cycles of mutation and screening.

In order to optimise the Box-A of HMGB1 protein and to obtain thepolypeptide variants of the present invention with higher stabilityagainst proteases, a particular Nautilus proprietary technology fordirected evolution has been applied. In particular, a so-calledtwo-dimensional rational mutagenesis scanning approach (“2-D scanning”)has been applied, which is described in the Nautilus patent applicationWO 2004/022593, said application being herein incorporated by reference.

Nautilus 2-D scanning approach for protein rational evolution is basedon a process, in which two dimensions of the target protein are scannedby serial mutagenesis in order to find the right amino acid change thatis needed at the right amino acid position. The first dimension that isscanned is the amino acid position along the target protein sequence, inorder to identify those specific amino acid residues to be replaced withdifferent amino acids. These amino acid positions are referred to asis-HIT target positions. The second dimension is the specific amino acidtype selected for replacing a particular is-HIT target position.According to a particular approach of the 2-D scanning method, a numberof target positions along the protein sequence are selected, in silico.As used herein, in silico refers to research and experiments performedusing a computer. In this context, in silico methods include, but arenot limited to, molecular modeling studies and biomolecular dockingexperiments. Therefore, the amino acid target positions on the proteinsequence are identified without use of experimental biological methods.Once a protein feature to be optimised is selected, diverse sources ofinformation or previous knowledge are exploited in order to determinethose amino acid positions that may be amenable to improve the protein'sfitness by replacement with a different amino acid. In particular the“is-HIT target positions” are identified based on three factors, being(i) the protein feature to be evolved and optimised, (ii) the protein'samino acid sequence and/or (iii) the known properties of the individualamino acids.

In the specific context of the present invention, the “in silico HITs”(“is-HITs”) are all possible candidate amino acid positions along thetarget protein's primary sequence that might be involved as target forthe proteolytic activity of proteases. Based on the specific list ofproteases considered in the context of the present invention (FIG. 1),the complete list of all amino acid sequences that could potentially betargeted within the wild type HMGB1 Box-A amino acid sequence isdetermined.

Once the is-HIT target positions have been selected, mutagenesis then isperformed by the replacement of single amino acid residues at thespecific acid target positions on the protein backbone. The mutagenesisis performed by residue replacement “one-by-one” in addressable arraysand molecules containing the preselected amino acid changes at each ofthe target amino acid positions are produced.

The choice of the replacing amino acid takes into account the need topreserve the physicochemical properties such as hydrophobicity, chargeand/or polarity of essential residues (such as catalytic and bindingresidues). Numerous methods of selecting replacing amino acids are wellknown in the art, in particular, amino acid substitution matrixes areused for this purpose. A very preferred technology according to thepresent invention makes use of the so-called “Percent Accepted Mutation”(PAM) (Dayhoff et al., Atlas of protein sequence and structure,5(3):345-352, 1978), as shown in FIG. 2. PAM values are used in order toselect an appropriate group of replacement amino acids. PAM values,originally developed to produce alignments between protein sequences,are available in the form of probability matrixes, which reflect anevolutionary distance. “Conservative substitutions” of a residue in areference sequence are those substitutions that are physically andfunctionally similar to the corresponding reference residues, e.g. thosethat have a similar size, shape, electric charge, chemical properties,including the ability to form covalent or hydrogen bonds, or the like.Preferred conservative substitutions show the highest scores fittingwith the PAM matrix criteria in the form of “accepted point mutations”.The PAM250 matrix is used in 2-D scanning to identify the replacingamino acids for the is-HITs in order to generate conservative mutationswithout affecting the protein function. At least, the two amino acidswith the highest values in PAM250 matrix, corresponding to “conservativesubstitutions” or “accepted point mutations”, are chosen. Thereplacement of amino acids by cysteine residues is explicitly avoided,since this change would potentially lead to the formation ofintermolecular disulfide bonds.

Using the above-resumed Nautilus Biotech directed evolution technology,the inventors of the present application were able to obtain polypeptidevariants of the HMGB1 Box-A which differ from the amino acid sequence ofthe native target polypeptide by one or more mutation.

In the context of the present invention, where reference is made to theterm “HMGB1 Box-A or amino acid sequence of HMGB1 Box-A”, it is referredto both human and non-human HMGB1 Box-A. In a preferred embodiment ofthe present invention, the systematic mutation of single amino acid onthe critical is-HITs positions for proteases has been obtained on thewild type of human HMGB1 Box-A protein and on the wild type of Anophelesgambia HMGB1 Box-A protein. The choice of the species Anopheles gambiawas made by the inventors of the present application after a properstructural and phylogenetic analysis showing a 68% identity and a 88%homology of the human and Anopheles HMGB1 Box-A.

“Biologically active fragments of HMGB1 Box-A” as used herein are meantto encompass parts of the known wild type HMGB1 Box-A protein, for whichat least one of the biological activities of the corresponding matureprotein is still observable when known tests are being used. Preferably,a fragment of the mature protein is considered as biologically active ifan antagonist activity with respect to the pro-inflammatory activity ofthe HMGB1 B-Box and the HMGB1 protein as a whole can be determined.Biologically active fragments of native HMGB1 Box-A are fragments of atleast 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75 or 80 amino acids.Preferred biologically active fragments of native HMGB1 Box-A used inthe context of the present invention comprises fragments of at least 77or of at least 54 amino acids, respectively.

The term “mutation” as used in the context of the present invention canbe understood as substitution, deletion and/or addition of single aminoacid in the target sequence. Preferably, the mutation of the targetsequence in the present invention is a substitution. The substitutioncan occur with different genetically encoded amino acid or bynon-genetically encoded amino acids. Examples for non-geneticallyencoded amino acids are homocystein, hydroxyproline, ornithin,hydroxylysine, citrulline, carnitine, etc.

The polypeptide variants of the present invention obtained by usingdirected evolution technology are mutant proteins which differ from theamino acid sequence of the wild type HMGB1 Box-A by the mutation of oneor more single amino acid. In a very preferred embodiment of the presentinvention, only one amino acid replacement occurs on the sequence of thenative protein. In this case, the polypeptide variant of the inventionis obtained by the modification of the native protein, such that theamino acid sequence of the variant differs from that of the nativeprotein by a single amino acid change at only one of the is-HIT targetpositions. It is, however, encompassed by the subject of the presentinvention that the native protein can be further optimised byreplacement of a plurality, e.g two or more, of is-HIT target positionson the same protein molecule. According to this variant of theinvention, polypeptide variants are obtained by combining the singlemutation into a single protein molecule. The modified polypeptidevariants having more single amino acid replacement can differ from thewild type protein sequence by amino acid replacements on 1-10,preferably 2, 3, 4, 5 and 6 different amino acid target positions.

The selection of the candidate lead of the series of polypeptidevariants produced with the technology used in the present invention isbased both on the more favourable pharmacokinetic profile, obtainedthanks to the longer resistance to proteases and on a betterpharmacodynamic profile thanks to an increased intrinsic activity andbinding affinity which gives a greater antagonistic activity than thenative HMGB1 Box-A protein.

In a particular embodiment of the invention, starting from Human HMGB1Box-A as starting native protein, three groups of polypeptide variantsare obtained. In particular, one group of polypeptide variants isderived from single mutations introduced into the full-length amino acidsequence (84 amino acids) from Human HMGB1 Box-A (SEQ ID NO:1). Theother two groups of inventive polypeptide variants are generatedstarting from biologically active fragments of Human HMGB1 Box-A of 77amino acids (SEQ ID NO:117) and 54 amino acids (SEQ ID NO:223),respectively.

In a further particular embodiment of the invention, starting fromAnopheles gambia HMGB1 Box-A as starting native protein, three groups ofpolypeptide variants are obtained. In particular, one group ofpolypeptide variants is derived from single mutations introduced intothe full-length amino acid sequence (84 amino acids) from Anophelesgambia HMGB1 Box-A (SEQ ID NO:301). The other two groups of inventivepolypeptide variants are generated starting from biologically activefragments of Anopheles gambia HMGB1 Box-A of 77 amino acids (SEQ IDNO:419) and 54 amino acids (SEQ ID NO:529), respectively.

Hence, the above-mentioned very preferred polypeptide variants of thisinvention are defined as below.

1) On the human HMGB1 Box-A full-length fragment of 84 amino acidsdefined by the sequence SEQ ID NO:1 (FIG. 3 a), 53 amino acid positions,recognized as substrate for different proteases (cf. FIG. 1), areidentified. The numbering corresponds to that in the wild type protein:

K2, D4, P5, K6, K7, P8, R9, K11, M12, Y15, F17, F18, R23, E24, E25, K27,K28, K29, P31, D32, F37, E39, F40, K42, K43, E46, R47, W48, K49, M51,K54, E55, K56, K58, F59, E60, D61, M62, K64, D66, K67, R69, Y70, E71,R72, E73, M74, K75, Y77, P79, P80, K81, E83.

The native amino acid at each of these positions is replaced by residuesdefined by the substitution matrix PAM250 (cf. FIG. 2). In particular,the performed residue substitutions are as listed below.

R to H, Q

E to H, Q, N

K to Q, T

D to N, Q

M to I, V

P to A, S

Y to I, H

F to I, V

W to Y, S

A total of 115 polypeptide variants of Box-A of human HMGB1 aregenerated (FIG. 3 a). These polypeptide variants are defined insequences SEQ ID NOs:2 to 116.

2) On the Human HMGB1 Box-A biologically active fragment of 77 aminoacids, defined in sequence SEQ ID NO:117 (FIG. 4 a), 48 amino acidpositions, recognized as substrate for different proteases (cf. FIG. 1),are identified. The numbering is in accordance to their position in SEQID NO:117:

P1, R2, K4, M5, Y8, F10, F11, R16, E17, E18, K20, K21, K22, P24, D25,F30, E32, F33, K35, K36, E39, R40, W41, K42, M44, K47, E48, K49, K51,F52, E53, D54, M55, K56, D59, K60, R62, Y63, E64, R65, E66, M67, K68,Y70, P72, P73, K74, E76.

The native amino acid in each of these positions is replaced by residuesdefined by the substitution matrix PAM250 (cf. FIG. 2). In particular,the performed residue substitutions are as listed below.

R to H, Q

E to H, Q, N

K to Q, T

D to N, Q

M to I, V

P to A, S

Y to I, H

F to I, V

W to Y, S

A total of 105 polypeptide variants of Box-A of human HMGB1 fragment of77 amino acids are generated (FIG. 4 b) and defined as in sequences SEQID NOs:118 to 222.

3) On the Human HMGB1 Box-A biologically active fragment of 54 aminoacids defined in sequence SEQ ID NO:223 (FIG. 5 a), 35 amino acidpositions, recognized as substrate for different proteases (FIG. 1), areidentified. The numbering is in accordance to their position in SEQ IDNO:223:

P1, D2, F7, E9, F10, K12, K13, E16, R17, W18, K19, M21, K24, E25, K26,K28, F29, E30, D31, M32, K34, D36, K37, R39, Y40, E41, R42, E43, M44,K45, Y47, P49, P50, K51, E53.

The native amino acid at each of these positions is replaced by residuesdefined by the substitution matrix PAM250 (cf. FIG. 2). In particular,the performed residue substitutions are as listed below.

R to H, Q

E to H, Q, N

K to Q, T

D to N, Q

M to I, V

P to A, S

Y to I, H

F to I, V

W to Y, S

A total of 77 polypeptide variants of Box-A of human HMGB1 fragment of54 amino acids are generated (FIG. 5 b) and defined as in sequences SEQID NOs:224 to 300.

4) On the Anopheles gambia (XP_(—)311154) HMGB1 Box-A full-lengthfragment of 84 amino acids, defined by the sequence SEQ ID NO:301 (FIG.6 a), 53 amino acid positions, recognized as substrate for differentproteases (FIG. 1), were identified. The numbering is in accordance withthe position in the native protein.

K2, K4, D5, K7, P8, R9, R11, M12, Y15, F17, F18, R23, E24, E25, K27,K28, K29, P31, E32, E33, F37, E39, F40, R42, K43, E46, R47, W48, K49,M51, L52, D53, K54, E55, K56, R58, F59, E61, M62, E64, K65, D66, K67,R69, Y70, E71, L72, E73, M74, Y77, P79, P80, K81.

The native amino acid at each of these positions was replaced byresidues defined by the substitution matrix PAM250 (cf. FIG. 2). Theperformed actual residue substitutions are as listed below.

R to H, Q

E to H, Q, N

K to Q, T

D to N, Q

M to I, V

P to A, S

Y to I, H

F to I, V

W to Y, S

A total of 117 variants of Box-A of HMGB1 Anopheles gambia(XP_(—)311154) were generated (FIG. 6 b) and identified in the sequencesas defined in SEQ ID NOs:302 to 418.

5) On the Anopheles gambia (XP_(—)311154) HMGB1 Box-A biologicallyactive fragment of 77 amino acids, defined in sequence SEQ ID NO:419(FIG. 7 a), 49 amino acid positions, recognized as substrate fordifferent proteases (cf. FIG. 1), were identified. The numbering is inaccordance with the position in the sequence as defined in SEQ IDNO:419.

P1, R2, R4, M5, Y8, F10, F11, R16, E17, E18, K20, K21, K22, P24, E25,E26, F30, E32, F33, R35, K36, E39, R40, W41, K42, M44, L45, D46, K47,E48, K49, R51, F52, E54, M55, E57, K58, D59, K60, R62, Y63, E64, L65,E66, M67, Y70, P72, P73, K74.

The native amino acid at each of these positions was replaced byresidues defined by the substitution matrix PAM250 (cf. FIG. 2). Theperformed actual residue substitutions are as listed below.

R to H, Q

E to H, Q, N

K to Q, T

D to N, Q

M to I, V

P to A, S

Y to I, H

F to I, V

W to Y, S

A total of 109 polypeptide variants of Box-A of HMGB1 fragment of 77amino acids were generated (FIG. 7 b) and identified as defined insequences SEQ ID NOs:420 to 528.

6) On the Anopheles gambia (XP_(—)311154) HMGB1 Box-A biologicallyactive fragment of 54 amino acids defined in sequence SEQ ID NO:529(FIG. 8 a), 36 amino acid positions, recognized as substrate fordifferent proteases (cf. FIG. 1), were identified. The numbering is inaccordance with the position on the sequence as defined in SEQ IDNO:529.

P1, E2, E3, F7, E9, F10, R12, K13, E16, R17, W18, K19, M21, L22, D23,K24, E25, K26, R28, F29, E31, M32, E34, K35, D36, K37, R39, Y40, E41,L42, E43, M44, Y47, P49, P50, K51.

The native amino acid in each of these positions was replaced byresidues defined by the substitution matrix PAM250 (cf. FIG. 2). Theperformed actual residue substitutions are as listed below.

R to H, Q

E to H, Q, N

K to Q, T

D to N, Q

M to I, V

P to A, S

Y to I, H

F to I, V

W to Y, S

A total of 81 polypeptide variants of Box-A of HMGB1 Anopheles gambia(XP_(—)311154) fragment of 54 amino acids were generated (FIG. 8 b) andidentified in the sequences as defined in SEQ ID NOs:530 to 610.

It is noted that the amino acids which occur in the various amino acidsequences appearing herein are identified according to their knownone-letter code abbreviations. It should be further noted that all aminoacid residue sequences represented herein by their one-letterabbreviation code have a left-to-right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus.

Accordingly, the present invention provides modified polypeptidevariants that exhibit increased resistance to the proteolytic activityof proteases and/or peptidases compared to the wild type HMGB1 Box-Aprotein together with a similar or even higher antagonistic activitythan the native HMGB1 Box-A protein. The polypeptide variants of theinvention in particular exhibit an increase in the resistance to theproteolytic activity of the human proteases and/or peptidases, inparticular of the human serum proteases and/or human gastro-intestinalproteases or peptidases. Preferred proteases are listed in FIG. 1. In amore preferred embodiment of the invention, polypeptide variants exhibitan increase in the resistance to the proteolytic activity of at least aprotease selected from the group comprising chymotrypsin, trypsin,endoprotease, endopeptidases or a combination thereof.

In particular, the resistance to proteolysis is at least 10%, 20%, 30%,40%, 50%, 70%, 80%, 90%, 95% or higher compared to the unmodified wildtype HMGB1 Box-A. Protease resistance was measured at differenttimepoints (between 5 minutes and 8 hours) at 25° C. after incubation of20 μg of Box-A wild type or variants with a mixture of proteases at 1%w/w of total proteins. The mixture of the proteases was prepared freshlyat each assay from stock solutions of endoproteinase Glu-C (SIGMA) 200μg/ml; trypsin (SIGMA) 400 μg/ml and α-chymotrypsin (SIGMA) 400 μg/ml.After protease incubation the reaction was stopped adding 10 μl ofanti-proteases solution (Roche) and the samples were stored at −20° C.for the biological activity assay.

In particular, in order to identify the most preferred polypeptidevariants of the invention, studies have been conducted to determine thevariants which show both a similar or even improved activity and anincreased protease resistance compared to the wild-type HMGB1 Box-Aprotein. For this purpose, the activity of Box-A polypeptide variants ofhuman HMGB1 Box-A full-length fragment in inhibiting HMGB1-inducedNIH/3T3 cell migration was determined in chemotaxis assays in comparisonto Box-A wild-type (Example 6 and FIGS. 12.1 to 12.9). Moreover for thepolypeptide variants, which show a similar or even higher antagonisticactivity than the native HMGB1 Box-A protein of SEQ ID NO:1 the in vitroresistance to protease digestion was determined by incubation of each ofthese polypeptide variants with a mixture of trypsin, α-chymotrypsin,endoproteinase Asp-N and endoproteinase Glu-C (sigma). This proteaseresistance test is described in Example 7 and the results of proteaseresistance profile of said variants are shown in FIGS. 14.1 to 14.67.

From the results it can be gathered that the preferred polypeptidevariants of the present invention are those variants which show asimilar or higher antagonistic activity together with an increasedprotease resistance. In particular, the preferred polypeptide variantsare the polypeptides of SEQ ID NOs: 33, 35, 37-39, 42-45, 47-49, 52, 55,57, 59, 62, 64, 67, 69 and 104. Among these preferred polypeptidevariants, the most preferred variants are those defined in SEQ IDNos:45, 49, 52, 55, 59, 64 and 67. These very preferred polypeptidevariants show a dramatically improved proteinase resistance profilecompared with the wild-type human HMGB1 Box-A of SEQ ID NO:1 (cf.results of Example 7).

As a consequence of the increased stability due to the increasedresistance to proteases activity, the polypeptide variants of thepresent invention also exhibit a longer half-life in body fluidscompared to the wild type HMGB1 Box-A. In particular, the half-life inserum and/or in blood is increased, whereby an increase of at least 10minutes, 20 minutes, 30 minutes, 60 minutes or even longer, compared tothe wild type HMGB1 Box-A is observed.

A further aspect of the present invention is a nucleic acid moleculeencoding a polypeptide variant of the present invention. In particular,the present invention refers to nucleic acid molecules encodingpolyeptide variants as defined in SEQ ID NO:2 to 116, 118 to 222, 224 to300, 302 to 418, 420 to 528 and 530 to 610. Even more preferably, thepresent invention refers to nucleic acid molecules encoding for thepreferred polypeptide variants as defined above.

A still further aspect of the present invention is a vector comprising anucleic acid molecule as defined above.

Furthermore, the present invention refers to a method for producing apolypeptide variant as described above comprising (i) introducing anucleic acid molecule as defined above into a host cell and (ii)culturing the cell, under conditions in which the encoded polypeptidevariant is expressed. Preferably the host cell is a mammalian, insect orbacterial cell, in particular E. Coli, preferably the M15 strain.

A further method for producing a polypeptide variant as described aboveis the use of chemical peptide synthesis, e.g. a solid phase peptidesynthesis according to standard methods.

The polypeptide variants of the present invention exhibit an increasedresistance to proteolysis and thus a higher stability compared to theunmodified wild type protein. Consequently, the peptides of theinvention also exhibit improved therapeutic and biological propertiesand activity. In fact, they show a more favorable pharmacokinetic andpharmacodynamic profile than native HMGB1 Box-A.

The invention is therefore directed to the use of the above-mentionedpolypeptide variants of HMGB1 Box-A, obtained through systematicmutations of single amino acids in the sequence of HMGB1 Box-A or of itsbiologically active fragments as active agent in a medicament. In apreferred embodiment, the present invention refers to the use of thepreferred polypeptide variants as defined above as active agent in amedicament.

A still further aspect of the invention is hence the use of theinventive polypeptide variants, in particular of the preferredpolypeptide variants of the present invention as defined above, for themanufacture of a medicament for the prevention and/or treatment ofextracellular HMGB1-associated pathologies or pathologies associatedwith the HMGB1 homologous proteins. In particular, the HMGB1 associatedpathologies are pathologies which are mediated by a multipleinflammatory cytokine cascade.

The broad spectrum of pathological conditions induced by theHMGB1-chemokine and by the HMGB1-induced cascade of inflammatorycytokines are grouped in the following categories: inflammatory disease,autoimmune disease, systemic inflammatory response syndrome, reperfusioninjury after organ transplantation, cardiovascular affections, obstetricand gynecologic disease, infectious (viral and bacterial) disease,allergic and atopic disease, solid and liquid tumor pathologies,transplant rejection diseases, congenital diseases, dermatologicaldiseases, neurological diseases, cachexia, renal diseases, iatrogenicintoxication conditions, metabolic and iodiopathic diseases.

HMGB1-associated pathologies according to the present invention arepreferably pathological conditions mediated by activation of theinflammatory cytokine cascade. Non limiting examples of conditions whichcan be usefully treated using the present invention include the broadspectrum of pathological conditions induced by the HMGB1-chemokine andby the HMGB1-induced cascade of inflammatory cytokines grouped in thefollowing categories: restenosis and other cardiovascular diseases,reperfusion injury, inflammation diseases such as inflammatory boweldisease, systemic inflammation response syndrome, e.g. sepsis, adultrespiratory distress syndrome, etc, autoimmune diseases such asrheumatoid arthritis and osteoarthritis, obstetric and gynaecologicaldiseases, infectious diseases, atopic diseases, such as asthma, eczema,etc, tumor pathologies, e.g. solid or non-solid tumor diseasesassociated with organ or tissue transplants, such as reperfusioninjuries after organ transplantation, organ rejection andgraft-versus-host disease, congenital diseases, dermatological diseasessuch as psoriasis or alopecia, neurological diseases, opthalmologicaldiseases, renal, metabolic or idiopathic diseases and intoxicationconditions, e.g. iatrogenic toxicity, wherein the above diseases arecaused by, associated with and/or accompanied by HMGB1 protein release.

In particular, the pathologies belonging to inflammatory and autoimmunediseases include rheumatoid arthritis/seronegative arthropathies,osteoarthritis, inflammatory bowel disease, Crohn's disease, intestinalinfarction, systemic lupus erythematosus, iridoeyelitis/uveitis, opticneuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,systemic sclerosis and scleroderma. Systemic sclerosis and sclerodermasystemic inflammatory response includes sepsis syndrome (including grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, septic conjunctivitis),meningococcemia, trauma hemorrhage, hums, ionizing radiation exposure,acute and chronic prostatitis, acute and chronic pancreatitis,appendicitis, peptic, gastric and duodenal ulcers, peritonitis,ulcerative, pseudomembranous, acute and ischemic cholitis,diverticulitis, achalasia, cholangitis, cholecystitis, enteritis, adultrespiratory distress syndrome (ARDS). Reperfusion injury includespost-pump syndrome and ischemia-reperfusion injury. Cardiovasculardisease includes cardiac stun syndrome, myocardial infarction andischemia, atherosclerosis, thrombophlebitis, endocarditis, pericarditis,congestive heart failure and restenosis. Obstetric and gynecologicdiseases include premature labour, endometriosis, miscarriage, vaginitisand infertility. Infectious diseases include HIV infection/HIVneuropathy, meningitis, B- and C-hepatitis, herpes simplex infection,septic arthritis, peritonitis, E. coli 0157:H7, pneumonia epiglottitis,haemolytic uremic syndrome/thrombolytic thrombocytopenic purpura,candidiasis, filariasis, amebiasis, malaria, Dengue hemorrhagic fever,leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis,gas gangrene, mycobacterium tuberculosis, mycobacterium aviumintracellulare, pneumocystis carinii pneumonia, pelvic inflammatorydisease, orchitis/epidydimitis, legionella, Lyme disease, influenza A,Epstein-Barr Virus, Cytomegalovirus, viral associated hemiaphagocyticsyndrome, viral encephalitis/aseptic meningitis. Allergic and atopicdisease include asthma, allergy, anaphylactic shock, immune complexdisease, hay fever, allergic rhinitis, eczema, allergic contactdermatitis, allergic conjunctivitis, hypersensitivity pneumonitis.Malignancies (liquid and solid tumor pathologies) include ALL, AML, CML,CLL, Hodgkin's disease, non Hodgkin's lymphoma, Kaposi's sarcoma,colorectal carcinoma, nasopharyngeal carcinoma, malignant histiocytosisand paraneoplastic syndrome/hypercalcemia of malignancy. Transplantdiseases include organ transplant rejection and graft-versus-hostdisease. Congenital disease includes cystic fibrosis, familialhematophagocytic lymphohistiocytosis and sickle cell anemia.Dermatologic disease includes psoriasis, psoriatic arthritis andalopecia. Neurologic disease includes neurodegenerative diseases(multiple sclerosis, migraine, headache, amyloid-associated pathologies,prion diseases/Creutzfeld-Jacob disease, Alzheimer and Parkinson'sdiseases, multiple sclerosis, amyotrophic emilateral sclerosis) andperipheral neuropathies, migraine, headache. Renal disease includesnephrotic syndrome, hemodialysis and uremia, latrogenic intoxicationcondition includes OKT3 therapy, Anti-CD3 therapy, Cytokine therapy,Chemotherapy, Radiation therapy and chronic salicylate intoxication.Metabolic and idiopathic disease includes Wilson's disease,hemochromatosis, alpha-1 antitrypsin deficiency, diabetes and diabetescomplications, weight loss, anorexia, cachexia, obesity, Hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation and primary biliary cirrhosis. Ophtalmological diseaseinclude glaucoma, retinopathies and dry-eye. A miscellanea of otherpathologies comprehends: multiple organ dysfunction syndrome, musculardystrophy, septic meningitis, atherosclerosis, epiglottitis, Whipple'sdisease, asthma, allergy, allergic rhinitis, organ necrosis, fever,septicaemia, endotoxic shock, hyperpyrexia, eosinophilic granuloma,granulomatosis, sarcoidosis, septic abortion, urethritis, emphysema,rhinitis, alveolitis, bronchiolitis, pharyngitis, epithelial barrierdysfunctions, pneumoultramicropicsilicovolcanoconiosis, pleurisy,sinusitis, influenza, respiratory syncytial virus infection,disseminated bacteremia, hydatid cyst, dermatomyositis, burns, sunburn,urticaria, warst, wheal, vasulitis, angiitis, myocarditis, arteritis,periarteritis nodosa, rheumatic fever, celiac disease, encephalitis,cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia,iatrogenic complications/peripheral nerve lesions, spinal cord injury,paralysis, uveitis, arthriditis, arthralgias, osteomyelitis, fasciitis,Paget's disease, gout, periodontal disease, synovitis, myastheniagravis, Goodpasture's syndrome, Babcets's syndrome, ankylosingspondylitis, Barger's disease, Retier's syndrome, bullous dermatitis(bullous pemphigoid), pemphigous and pemphigous vulgaris and alopecia.

In a further aspect of the invention, the use of the polypeptidevariants obtained through systematic mutations of amino acid sequencesof human and non-human Box-A of HMGB1, or of its biologically relevantfragments described above, is in combination with a further agent.

The further agent is preferably an agent capable of inhibiting an earlymediator of the inflammatory cytokine cascade. Preferably, this furtheragent is an antagonist or inhibitor of a cytokine selected from thegroup consisting of TNF, IL-1α, IL-1β, IL-Ra, IL-6, IL-8, IL-10, IL 13,IL-18, IFN-γ MIP-1α, MIF-1β, MIP-2, MIF and PAF.

The further agent used in combination with the polypeptide variants ofHMGB1 Box-A, or of its biologically relevant fragments, may also be aninhibitor of RAGE, e.g. an antibody directed to RAGE, a nucleic acid ornucleic acid analogue capable of inhibiting RAGE expression, e.g. anantisense molecule, a ribozyme or a RNA interference molecule, or asmall synthetic molecule antagonist of the interaction of HMGB1 withRAGE, preferably of the interaction of the non-acetylated or/andacetylated form of HMGB1 with RAGE, or soluble RAGE (sRAGE). Theantibody to RAGE is preferably a monoclonal antibody, more preferably achimeric or humanised antibody or a recombinant antibody, such as asingle chain antibody or an antigen-binding fragment of such anantibody. The soluble RAGE analog may be optionally present as a fusionprotein, e.g. with the Fc domain of a human antibody. The smallsynthetic molecular antagonist of the HMGB1 interaction with RAGEpreferably has a molecular weight of less than 1000 Dalton. The smallsynthetic molecular antagonist preferably inhibits the interaction ofRAGE with the non-acetylated form or/and with the acetylated form ofHMGB1 and with the non-acetylated form or/and with the acetylated formof HMGB1 homologous proteins, particularly HMGB2, HMGB3, HMG-1L10,HMG-4L or/and SP100-HMG.

The further agent used in combination with the polypeptide variants ofHMGB1 Box-A, or of its biologically relevant fragments, may also be aninhibitor of the interaction of a Toll-like receptor (TLR), e.g. ofTLR2, TLR4, TLR7, TLR8 or/and TLR9, with HMGB1, which inhibitor ispreferably a monoclonal or polyclonal antibody, a nucleic acid ornucleic acid analogue capable of inhibiting TLR expression, e.g. anantisense molecule, a ribozyme or a RNA interference molecule, or asynthetic molecule preferably having a size of less than 1000 Dalton.The inhibitor may be a known inhibitor of a Toll-like receptor, inparticular of TLR2, TLR4, TLR7, TLR8 or/and TLR9. The inhibitorpreferably inhibits the interaction of the Toll-like receptor with thenon-acetylated form or/and the acetylated form of HMGB1 and with thenon-acetylated form or/and with the acetylated form of HMGB1 homologousproteins, in particular HMGB2, HMGB3, HMG-1L10, HMG4L or/and SP100-HMG.

In still another embodiment, the further agent used in combination withthe polypeptide variants of HMGB1 Box-A, or of its biologically relevantfragments, is the functional N-terminal lectin-like domain (D1) ofthrombomodulin. The D1 domain of thrombomodulin is able to intercept thenon-acetylated form and/or the acetylated form of released HMGB1 and ofreleased HMGB1 homologous proteins, in particular HMGB2, HMGB3,HMG-1L10, HMG-4L or/and SP100-HMG, preventing thus their interactionwith RAGE and Toll-like receptors. The D1 domain of thrombomodulin maybe native or mutated in order to make it resistant to proteases.

The further agent may also be a synthetic double-stranded nucleic acidor nucleic acid analogue molecule with a bent shape structure,particularly a double-stranded bent DNA, PNA or DNA/PNA chimera orhybrid or a double-stranded cruciform DNA, PNA or DNA/PNA chimera orhybrid structure, capable of binding to the HMGB1 protein. Preferrednucleic acids and nucleic analogue molecules are disclosed in a co-ownedand co-pending international patent application No. PCT/EP2005/007198filed on 4 Jul. 2005 (claiming the priority of U.S. provisionalapplication No. 60/584,678 filed on 2 Jul. 2004), which are incorporatedherein by reference. The synthetic double-stranded nucleic acid ornucleic acid analogue molecule with a bent shape structure is preferablycapable of binding to the non-acetylated or/and to the acetylated formof HMGB1 and the non-acetylated or/and the acetylated form of HMGB1homologous proteins, in particular HMGB2, HMGB3, HMG-1 L10, HMG4L or/andSP100-HMG.

In a still further embodiment, the further agent used in combinationwith the inventive polypeptide variants is K-252a or/and a salt orderivative thereof or a polymer conjugate of K-252a or/and of aderivative thereof. The use of K-252a or polymer conjugate of K-252a andderivatives thereof is disclosed in a co-owned and co-pendinginternational patent application No. PCT/EP2005/008258 and USprovisional application filed on 25 Aug. 2005.

Therefore, a further aspect of the present invention is a pharmaceuticalcomposition comprising an effective amount of at least one of thepolypeptide variants of HMGB1 Box-A or a biologically active fragmentthereof in particular of the preferred polypeptide variants of theinvention as defined above as an active ingredient for the treatment ofHMGB1-associated pathologies and pharmaceutically acceptable carriers,diluents and/or adjuvants. The pharmaceutical composition of the presentinvention is preferably suitable for the treatment of pathologiesassociated with the non-acetylated or/and the acetylated form of HMGB1and/or of HMGB1 homologous proteins. In a further preferred embodiment,the pharmaceutical composition of the present invention comprising theat least one polypeptide variant also comprises a further agent asdefined above. The pharmaceutical composition of the present inventionmay be used for diagnostic or for therapeutic applications.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's conditions.Administration may be achieved in a single dose or repeated doses atintervals. Dosage amount and interval may be adjusted individually inorder to provide the therapeutical effect which results in ameliorationof symptoms or a prolongation of the survival in a patient. The actualamount of composition administered will, of course, be dependent on thesubject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgement of theprescribing physician. A suitable daily dosage will be between 0.001 to10 mg/kg, particularly 0.1 to 5 mg/kg.

The administration may be carried out by known methods, e.g. byinjection, in particular by intravenous, intramuscular, transmucosal,subcutaneous or intraperitoneal injection and/or by oral, topical,nasal, inhalation, aerosol and/or rectal application, etc. Theadministration may be local or systemic.

In addition, the variants of Box-A of HMGB1, or of its pharmacologicallyactive fragments, object of this invention can be reversibly immobilizedand/or adsorbed on the surface and/or inside medical devices or drugrelease/vehicling systems (microspheres). Medical devices andmicrospheres can be reversibly loaded with the polypeptide variants ofBox-A object of this invention, through their binding, impregnationand/or adsorption on the surface of the medical device or of themicrosphere or on a layer that coats its surface. When the medicaldevice or the microsphere come into contact with biological fluids, thereversibly immobilized variant of Box-A is released. Therefore, themedical device and the microsphere act as drug-releasing tools thatelute the molecule object of this invention in such a way that theirrelease kinetics can be controlled, ensuring controlled or sustainedrelease, as required by the treatment. The methods forcoating/impregnating the medical devices and loading microspheres arewell known by experts in these technologies.

Thus, a further aspect of this invention is the way of using thevariants of Box-A of HMGB1 or its pharmacologically relevant fragments,wherein the mutated polypeptide molecules are reversibly immobilized onthe surface of medical devices or of microspheres or are adsorbed withinthem. These medical instruments are preferably surgical tools, implants,catheters or stents, for example stents for angioplasty and, inparticular, medicated drug-eluting stents.

Another aspect of the invention concerns a medical device reversiblycoated with at least one polypeptide variant of the invention. Such adevice can be selected from surgical instruments, implants, catheters orstents. Such a device may be useful for angioplasty.

The invention is further illustrated by the following Figures andExamples. The examples are intended to exemplify generic processes andare included for illustrative purpose only, without intention oflimiting the scope of the present invention.

FIG. 1 shows the proteases used for the in silico identification of theamino acid positions (is-HITs) on the HMGB1 Box-A amino acid sequencewhich are targets for the proteolytic activity.

FIG. 2 depicts the “Percent Accepted Mutation” (PAM 250) matrix. Valuesgiven to identical residues are shown in grey square. Highest values inthe matrix are shown in black square and correspond to the highestoccurrence of substitution between two residues.

FIG. 3 a displays the amino acid sequence of the native Human HMGB1Box-A made of 84 amino acid residues. In bold, the amino acids sensitiveto proteases proteolysis are identified, showing the is-HIT residuepositions.

FIG. 3 b shows the type of replacing amino acids on the respectiveis-HITs target positions selected to generate the polypeptide variant ofthe full-length human HMGB1 Box-A. Further, the specific amino acidsequences of the generated polypeptide variant are displayed in SEQ IDNOs:2 to 116.

FIG. 4 a displays the amino acid sequence of the biologically activefragment of Human HMGB1 Box-A made of 77 amino acid residues. In bold,the amino acids sensitive to proteases proteolysis are identified,showing the is-HIT residue positions.

FIG. 4 b shows the type of replacing amino acids on the respectiveis-HITs target positions selected to generate the polypeptide variant ofthe biologically active fragment of Human HMGB1 Box-A made of 77 aminoacid residues. Further the specific amino acid sequences of thegenerated polypeptide variant are displayed in SEQ ID NOs: 118 to 222.

FIG. 5 a displays the amino acid sequence of the biologically activefragment of Human HMGB1 Box-A made of 54 amino acid residues. In bold,the amino acids sensitive to proteases proteolysis are identified,showing the is-HIT residue positions.

FIG. 5 b shows the type of replacing amino acids on the respectiveis-HITs target positions selected to generate the polypeptide variant ofthe biologically active fragment of Human HMGB1 Box-A made of 54 aminoacid residues. Further, the specific amino acid sequences of thegenerated polypeptide variant are displayed in SEQ ID NOs: 224 to 300.

FIG. 6 a displays the amino acid sequence of the native Anopheles gambiaHMGB1 Box-A made of 84 amino acid residues. In bold, the amino acidssensitive to proteases proteolysis are identified, showing the is-HITresidue positions.

FIG. 6 b shows the type of replacing amino acids on the respectiveis-HITs target positions selected to generate the polypeptide variant ofthe full-length Anopheles gambia HMGB1 Box-A. Further, the specificamino acid sequences of the generated polypeptide variant are displayedin SEQ ID NOs: 302 to 418.

FIG. 7 a displays the amino acid sequence of the biologically activefragment of Anopheles gambia HMGB1 Box-A made of 77 amino acid residues.In bold, the amino acids sensitive to proteases proteolysis areidentified, showing the is-HIT residue positions.

FIG. 7 b shows the type of replacing amino acids on the respectiveis-HITs target positions selected to generate the polypeptide variant ofthe biologically active fragment of Anopheles gambia HMGB1 Box-A made of77 amino acid residues. Further the specific amino acid sequences of thegenerated polypeptide variant are displayed in SEQ ID NOs: 420 to 528.

FIG. 8 a displays the amino acid sequence of the biologically activefragment of Anopheles gambia HMGB1 Box-A made of 54 amino acid residues.In bold, the amino acids sensitive to proteases proteolysis areidentified, showing the is-HIT residue positions.

FIG. 8 b shows the type of replacing amino acids on the respectiveis-HITs target positions selected to generate the polypeptide variant ofthe biologically active fragment of Anopheles gambia HMGB1 Box-A made of54 amino acid residues. Further, the specific amino acid sequences ofthe generated polypeptide variant are displayed in SEQ ID NOs: 530 to610.

FIG. 9 shows the plasmid vector containing the nucleic acid sequenceencoding for the polypeptide variant of the present invention. Theplasmid contains the gene encoding for the polypeptide variant of thepresent invention, which is under control of the IPTG inducible T5promoter. The plasmid further contains an ampicillin resistant gene, a6×His-tag and several restriction sites.

FIG. 10 shows a graph displaying the correlation between the TNF-alpharelease induced by the stimulation of HMGB1 in RAW 264.7 cells.

FIG. 11 displays a dose-dependent inhibition of HMGB1-induced TNF-alpharelease by a Box-A His-tagged protein.

Figures and Tables 12.1 to 12.9 show the results of the chemotaxis assaydescribed of Example 6 performed on the HMGB1 Box-A polypeptide variantsof SEQ ID NO:2 to SEQ ID NO:116 of the present invention. In each figurethe activity of a set of polypeptide variants is tested compared to theactivity of human wild-type HMGB1 Box-A full-length fragment of SEQ IDNO:1 in the inhibition of HMGB1 induced NIH/3T3 cell migration. Eachfigure shows a table reporting the statistical analysis numerical dataand a column bar graph showing the chemotaxis assay results.

FIG. 12.1 and Table 12.1 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID NO:2 to 15 (identified in the Table andFigure with the code CT501, CT568, CT569, CT570, CT571, CT502, CT572,CT503, CT573, CT504, CT574, CT575, CT576 and CT505, respectively).

FIG. 12.2 and Table 12.2 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID NOs:16-23 and 25-29 (identified in theTable and Figure with the code CT577, CT578, CT506, CT579, CT580, CT581,CT507, CT582, CT584, CT508, CT509, CT510 and CT585, respectively).

FIG. 12.3 and Table 12.3 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID Nos:30-35 and 37-43 (identified in theTable and Figure with the code CT511, CT512, CT513, CT514, CT586, CT515,CT516, CT517, CT518, CT519, CT520, CT521 and CT522, respectively).

FIG. 12.4 and Table 12.4 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NOs:44-57(identified in the Table and Figure with the code CT523, CT524, CT525,CT526, CT527, CT528, CT588, CT529, CT530, CT589, CT590, CT531, CT591 andCT532, respectively).

FIG. 12.5 and Table 12.5 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID Nos:58-67 and 69-71 (identified in theTable and Figure with the code CT592, CT533, CT593, CT534, CT535, CT536,CT537, CT594, CT538, CT539, CT540, CT541 and CT542, respectively).

FIG. 12.6 and Table 12.6 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID Nos:72-85 (identified in the Table andFigure with the code CT596, CT597, CT598, CT599, CT600, CT601, CT602,CT603, CT543, CT544, CT545, CT546, CT547 and CT604, respectively).

FIG. 12.7 and Table 12.7 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID NOs:86-99 (identified in the Table andFigure with the code CT548, CT549, CT605, CT606, CT607, CT608, CT609,CT610, CT550, CT551, CT611, CT552, CT553 and CT554, respectively).

FIG. 12.8 and Table 12.8 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID Nos:100-113 (identified in the Table andFigure with the code CT555, CT556, CT557, CT558, CT559, CT612, CT560,CT561, CT613, CT562, CT563, CT564, CT565 and CT566, respectively).

FIG. 12.9 and Table 12.9 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID Nos:114-116 (identified in the Table andFigure with the code CT567, CT614 and CT615, respectively).

FIG. 13 shows the image of the Tricine SDS-PAGE gel loaded with humanHMGB1 Box-A wild type of SEQ ID NO:1 (CT500) at different timepointsafter protease digestion of the protease resistance testing described inExample 7. The Box-A wild type protein tested for protease resistance isa His-tagged protein. After 5 minutes of digestion CT500 shows two majorbands, one corresponding to the original protein in the sample and thesecond corresponding to the 84-aminoacid protein without the N-termHis-tag (indicated on the figure with an arrow). The profile of thissecond band shows resistance to proteases for 30 minutes. Minor bandspresent on this and other gels of FIG. 14.1 to FIG. 14.67 correspond toBox-A digested fragments.

FIG. 14.1 to FIG. 14.67 show the image of the Tricine SDS-PAGE gelloaded with the polypeptide variants of the present invention atdifferent timepoints after protease digestion of the protease resistancetesting described in Example 7. Box-A polypeptide variants tested forprotease resistance are Hig-tagged proteins. After 5 minutes ofdigestion the SDS-PAGE gel image of the polypeptide variants show twomajor bands, one corresponding to the original protein variant in thesample and the second corresponding to the Box-A 84 amino acid proteinvariant without the N-term His-tag.

FIG. 14.1 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:2 (CT501) at different timepoints after protease digestion.

FIG. 14.2 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:7 (CT502) at different timepoints after protease digestion.

FIG. 14.3 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:9 (CT503) at different timepoints after protease digestion.

FIG. 14.4 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:11 (CT504) at different timepoints after protease digestion.

FIG. 14.5 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:15 (CT505) at different timepoints after protease digestion.

FIG. 14.6 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:18 (CT506) at different timepoints after protease digestion.

FIG. 14.7: Tricine SDS-PAGE of the polypeptide variant of SEQ ID NO:22(CT507) at different timepoints after protease digestion.

FIG. 14.8 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:26 (CT508) at different timepoints after protease digestion.

FIG. 14.9 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:27 (CT509) at different timepoints after protease digestion.

FIG. 14.10 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:28 (CT510) at different timepoints after protease digestion.

FIG. 14.11 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:30 (CT511) at different timepoints after protease digestion.

FIG. 14.12 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:31 (CT512) at different timepoints after protease digestion.

FIG. 14.13 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:32 (CT513) at different timepoints after protease digestion.

FIG. 14.14 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:33 (CT514) at different timepoints after protease digestion.

FIG. 14.15 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:35 (CT515) at different timepoints after protease digestion.

FIG. 14.16 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:37 (CT516) at different timepoints after protease digestion.

FIG. 14.17 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:38 (CT517) at different timepoints after protease digestion.

FIG. 14.18 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:39 (CT518) at different timepoints after protease digestion.

FIG. 14.19 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:40 (CT519) at different timepoints after protease digestion.

FIG. 14.20 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:41 (CT520) at different timepoints after protease digestion.

FIG. 14.21 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:42 (CT521) at different timepoints after protease digestion.

FIG. 14.22 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:43 (CT522) at different timepoints after protease digestion.

FIG. 14.23 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:44 (CT523) at different timepoints after protease digestion.

FIG. 14.24 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:45 (CT524) at different timepoints after protease digestion.

FIG. 14.25 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:46 (CT525) at different timepoints after protease digestion.

FIG. 14.26 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:47 (CT526) at different timepoints after protease digestion.

FIG. 14.27 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:48 (CT527) at different timepoints after protease digestion.

FIG. 14.28 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:49 (CT528) at different timepoints after protease digestion.

FIG. 14.29 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:51 (CT529) at different timepoints after protease digestion.

FIG. 14.30 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:52 (CT530) at different timepoints after protease digestion.

FIG. 14.31 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:55 (CT531) at different timepoints after protease digestion.

FIG. 14.32 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:57 (CT532) at different timepoints after protease digestion.

FIG. 14.33 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:59 (CT533) at different timepoints after protease digestion.

FIG. 14.34 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:61 (CT534) at different timepoints after protease digestion.

FIG. 14.35 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:62 (CT535) at different timepoints after protease digestion.

FIG. 14.36 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:63 (CT536) at different timepoints after protease digestion.

FIG. 14.37 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:64 (CT537) at different timepoints after protease digestion.

FIG. 14.38 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:66 (CT538) at different timepoints after protease digestion.

FIG. 14.39 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:67 (CT539) at different timepoints after protease digestion.

FIG. 14.40 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:69 (CT540) at different timepoints after protease digestion.

FIG. 14.41 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:70 (CT541) at different timepoints after protease digestion.

FIG. 14.42 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:71 (CT542) at different timepoints after protease digestion.

FIG. 14.43 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:80 (CT543) at different timepoints after protease digestion.

FIG. 14.44 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:81 (CT544) at different timepoints after protease digestion.

FIG. 14.45 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:82 (CT545) at different timepoints after protease digestion.

FIG. 14.46 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:83 (CT546) at different timepoints after protease digestion.

FIG. 14.47 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:84 (CT547) at different timepoints after protease digestion.

FIG. 14.48 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:86 (CT548) at different timepoints after protease digestion.

FIG. 14.49 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:87 (CT549) at different timepoints after protease digestion.

FIG. 14.50 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:94 (CT550) at different timepoints after protease digestion.

FIG. 14.51 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:95 (CT551) at different timepoints after protease digestion.

FIG. 14.52 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:97 (CT552) at different timepoints after protease digestion.

FIG. 14.53 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:98 (CT553) at different timepoints after protease digestion.

FIG. 14.54 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:99 (CT554) at different timepoints after protease digestion.

FIG. 14.55 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:100 (CT555) at different timepoints after protease digestion.

FIG. 14.56 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:101 (CT556) at different timepoints after protease digestion.

FIG. 14.57 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:102 (CT557) at different timepoints after protease digestion.

FIG. 14.58 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:103 (CT558) at different timepoints after protease digestion.

FIG. 14.59 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:104 (CT559) at different timepoints after protease digestion.

FIG. 14.60 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:106 (CT560) at different timepoints after protease digestion.

FIG. 14.61 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:107 (CT561) at different timepoints after protease digestion.

FIG. 14.62 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:109 (CT562) at different timepoints after protease digestion.

FIG. 14.63 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:110 (CT563) at different timepoints after protease digestion.

FIG. 14.64 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:111 (CT564) at different timepoints after protease digestion.

FIG. 14.65 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:112 (CT565) at different timepoints after protease digestion.

FIG. 14.66 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:113 (CT566) at different timepoints after protease digestion.

FIG. 14.67 shows the Tricine SDS-PAGE of the polypeptide variant of SEQID NO:114 (CT567) at different timepoints after protease digestion.

FIG. 15 shows a table in which the results of the Tricine SDS-PAGE aresummarized. A cross indicates the presence on the gel of the bandcorresponding to the 84 amino acid long protein fragment of the HMGB1Box-A wild-type or of the HMGB1 Box-A polypeptide variant.

EXAMPLES 1. Production of HMGB1 Box-A Native and Variants in Bacteria

The in silico generated variants of HMGB1 Box-A were cloned from HMGB1protein into an inducible plasmid vector (FIG. 9) used to transform E.coli M15 strain competent cells. M15 cells were grown overnight in 1 mLof LB medium containing Kanamicyn and Ampicillin in 96 deep-well platesunder agitation (750 rpm). At OD_(600 nm) of 0.2-0.3 the cultures werediluted in 5 mL of LB medium in 24-well plates to reach an OD_(600 nm)of 0.07.

The M15 cells were incubated at 37° C. under constant agitation (200rpm). The production of Box-A (native or variants) was induced by theaddition of IPTG (1 mM final concentration) at OD_(600 nm) of 0.6. Theculture was continued for three hours at 37° C. under agitation (200rpm). M15 cells were then harvested by centrifugation at 1000 g for 15minutes, the supernatant was discarded and the pellet stored at −80° C.at least for 1 hour before cells lysis and Box-A purification.

2. Purification of HMGB1 Box-A Native and Variants

M15 cells pellet was thawed on ice for 15 min. The cells wereresuspended in 1 mL NPI-10 buffer containing 1 mg/mL Lysozyme andincubated for 30 min at RT under agitation at 750 rpm on a plate shaker.After the equilibration of Ni-NTA QIAfilter with 200 μL of Superflowresin (QIAGEN catalog #969261) and 600 μL of NPI-10 buffer the bacteriallysate was loaded and 200 μL of absolute EtOH added. Four wash stepswith 1 mL of NPI-20 were performed. The second and third washes weredone with 1 mL NPI-20 added with 100 μg/mL Polymyxin (Fluka catalog#81271) in order to deplete LPS contaminants. After wash steps Box-Anative and variants were eluted with 450 μL NPI-250. The samples werestored at 4° C.

Box-A native and variants were re-purified with a DetoxiGel polymyxincolumn (PIERCE) at 4° C. according to the supplier instructions. Finallythe eluted proteins were filtered (0.22 μm) in PBS and stored at 4° C.to be tested.

3. Box-A Biological Activity Assay

HMGB1 stimulates the secretion of TNF-alpha and of other cytokines aswell as the proliferation of macrophages and monocytes. Box-A acts as anantagonist by inhibiting the activity of HMGB1.

The activity of Box-A native and variants produced were measured by thelevel of inhibition on the stimulation produced by HMGB1 on RAW 264.7cells (murine macrophages, ATCC).

HMGB1 Box-A native and variants produced as described above were testedin a two-step process of screening directed to test i) their inhibitionof HMGB1 induced TNF-alpha release and ii) their resistance toproteolysis.

In order to determine the proper HMGB1 concentration to be used ininhibition assay RAW 264.7 cells were seeded in 96 well plates (4×10⁵cells/well) and grown overnight in RPMI 1640 medium supplemented with0.1% BSA. After overnight culture, cells were stimulated with HMGB1 (twotimes serial dilution concentrations between 100 μg/mL and 0.05 μg/mL)for 24 hours. The level of TNF-alpha produced was measured from cellmedia using ELISA (R&D systems), according to the manufacturerinstructions. As presented in FIG. 10, HMGB1 significantly stimulatedTNF-alpha release in macrophage cultures.

4. Box-A Inhibition of HMGB1 TNF-Alpha Release as Screening Test

Murine macrophage-like RAW 264.7 cells were seeded in 96 well plates(4×10⁵ cells/well) and grown overnight in RPMI 1640 medium supplementedwith 0.1% BSA. After overnight culture, cells were stimulated with anadequate concentration of HMGB1 and Box-A native or variants orHis-tagged (two times serial dilution between 20 μg/mL and 0.5 μg/mL)for 24 hours. The level of TNF-alpha was measured from cell media usingELISA (R&D systems), according to the manufacturer instructions.

FIG. 11 shows an example of dose-dependent inhibition of HMGB1 inducedTNF release by Box-A, with an EC50 of 7.5 μg/ml (solid line). 100%inhibition of TNF-alpha release is obtained with a concentration of 20μg/ml of Box-A. In parallel, TNF-alpha levels are measured in Box-Astimulated cells without HMGB1 in order to determine the presence orabsence of contaminating endotoxin in Box-A preparation and quantify anynon-HMGB1 dependent release of TNF-alpha. No release of TNF-alpha isobserved at all concentrations of Box-A used in the assay (dashed line).

5. Resistance to Proteolysis of Box-A Variants

Resistance of Box-A variants to proteolysis is determined as theresidual biological activity (in the HMGB1/RAW cells system) followingexposure to a mixture of selected proteases at increasing times ofincubation.

20 μg of Box-A native or variants were treated with a mixture ofproteases at 1% w/w of total proteins. The mixture of proteases wasfreshly prepared for each assay from stock solutions of endoproteinaseGlu-C (SIGMA; 200 μg/ml), trypsin (SIGMA; 400 μg/ml) and α-chymotrypsin(SIGMA; 400 μg/ml).

Samples were collected at different time points between 5 minutes and 8hours of incubation with proteases after stopping the reaction with theaddition of 10 μl of anti-proteases solution (Roche). Biologicalactivity of each sample was then evaluated by the screening testdescribed above in order to assess the residual activity at each timepoint.

6. In Vitro Activity Testing: NIH/373 Cell Migration Assay

The purpose of the present study was to evaluate the activity of each ofthe HMGB1 Box-A polypeptide variants of the human HMGB1 Box-Afull-length fragment as defined in SEQ ID NOs:2-116 and to compare theiractivity to that of human wild type HMGB1 Box-A full-length fragment ofSEQ ID NO:1 in order to select all the variants with similar or betteractivity than wild type. HMGB1 Box-A activity is evaluated in vitro asinhibition of HMGB1-induced NIH/3T3 cells migration.

6.1 Materials

HMGB1 Box-A wild type and variants (Nautilus Biotech)

NIH/3T3 cells (ATCC n. CRL-1658)

D-MEM medium (GIBCO; cat. n. 31966-021)

Foetal Bovine Serum (GIBCO; cat. n. 10270-106)

Penicillin-Streptomycin 10,000 U/ml (GIBCO; cat. n. 15140-122)

L-Glutamine 200 mM (GIBCO; cat. n. 25030-024)

TrypLE Select (GIBCO; cat. n. 12563-011)

Phosphate Buffered Saline (0.138 M NaCl, 0.0027 M KCl, 0.01 M phosphate,pH 7.4)

PVP free filters (8 μm pore size; 13 mm total diameter) (Neuro Probe;cat. n. PFA8)

Human fibronectin (Roche; cat. n. 1080938)

Blind Well Chemotaxis Chambers (Neuro Probe; cat. n. BW25)

GIEMSA Stain Modified (Sigma; cat. n. GS1L)

6.2 Filters Preparation

Polycarbonate membranes PVP free filters (8 μm pore size, 13 mm totaldiameter) are prepared about one hour before performing the experimentby coating them with 30 μl/filter of a solution 50 μg/ml of fibronectindispensed on the opaque side of the filter. The stock fibronectinsolution is prepared by diluting the lyophilized fibronectin in ddH₂O toa final concentration of 1 mg/ml and by keeping the solution about 1hour at 37° C. for complete dissolution. This stock solution can bestored at −20° C.

The filters are then left to dry under the laminar flux of the hood(about one hour).

6.3 Cells Preparation

NIH/3T3 cells are seeded the day before the experiment (approximately22-24 hours before performing the experiment) 10⁶ cells/plate.

When the filters are ready to use, the cells are detached with Trypsin,counted and resuspended 10⁶ cells/ml in serum free culture medium.

6.4 Chemotaxis Assay

In each chemotaxis experiment 14 different polypeptide variants of thehuman HMGB1 Box-A full-length fragment of the invention are tested.Growth cell medium without serum addition (w/o FBS) is used as negativecontrol representing spontaneous migration.

1 nM HMGB1 is used as positive control. HMGB1 Box-A wild type or thetested polypeptide variants 0.511 nM are added to 1 nM HMGB1 to inhibitHMGB1-induced NIH/3T3 cell migration.

Negative control (w/o FBS) and positive control (1 nM HMGB1) are testedin triplicate in each experiment.

HMGB1 Box-A wild type (SEQ ID NO:1) activity in inhibiting HMGB1-inducedcell migration is tested in triplicate in each experiment.

Each of the HMGB1 Box-A polypeptide (SEQ ID NO:2 to 116) variants istested in duplicate.

Blind Well Chemotaxis Chambers are used. The clean, dry lower well ofeach chamber is filled with 50 μl of DMEM without FBS added with theappropriate chemotactic agent and inhibitors. A slight positive meniscusshould form when the well is filled; this helps prevent air bubbles frombeing trapped when the filter is applied. With small forceps the filteris placed over the filled well (fibronectin treated side up), beingcareful not to trap air bubbles and not to touch the filter withfingers. The filter retainer is screwed in by hand. Cell suspension(50000 cells/50 μl) is pipetted into the upper well and 150 μl of serumfree medium are added to fill the upper well of the chamber. The filledchamber is incubated for 3 hours (37° C., 5% CO₂) to allow cellmigration. After incubation the fluid is removed from the filter. Theretainer is unscrewed and immersed in cool distilled water. The filteris lifted out with forceps, placed on a clean surface (solid paraffin)(migrated cells side up) and fixed with a needle (placed on the borderarea).

6.5 GIEMSA Staining of Migrated Cells

The filters are fixed with ethanol once and then washed three timesunder running water. A working solution of GIEMSA Stain Modified diluted1:10 in ddH₂O is prepared just before use. After washing of the filters,the staining is added and left to incubate for 20 minutes. Washing ofthe staining is performed under running water. The filters are thenplaced on slides with the migrated cells side down, and the non-migratedcells side is gently wiped off with a wet cotton swab (wipe twice, usingtwo swabs or both ends of a double-tipped swab) being careful not tomove the filter. After cleaning, a cover slide is placed on the filterand cells are counted under a microscope at 40× in 10 randomfields/filter.

6.6 Data Representation and Statistical Analysis

The results of the NIH/3T3 migration assay performed are reported in thetables and bar graphs shown in Figure and Table 12.1 to Figure and Table12.9.

Data are represented in bar columns as MEAN ±95% Cl.

One-way ANOVA followed by Dunnett's post test (control column data: 1 nMHMGB1 sample+HMGB1 Box-A WT sample) is the statistical analysisperformed.

When evaluating the results data, HMGB1 Box-A variants data having apost test p value<0.05 are considered significantly different from HMGB1Box-A wild type. If the mean of the Box-A polypeptide variant is higherthan that of Box-A wild type the column is coloured in red in the graphof the experiment shown in FIGS. 12.1 to 12.9. Those red columnsrepresent HMGB1 Box-A polypeptide variants showing less activity thanwild type in inhibiting HMGB1-induced cell migration.

If the mean of the polypeptide variant results lower than that of wildtype Box-A then the column is coloured in light blue in the graph of theexperiment shown in FIGS. 12.1 to 12.9. Those variants represent HMGB1Box-A variants showing higher activity than HMGB1 Box-A wild type ininhibiting HMGB1-induced cell migration.

HMGB1 Box-A variants data having a post test p value>0.05 are considerednot significantly different from HMGB1 Box-A wild type. The bar columnof those variants are coloured in green. Those variants represent HMGB1Box-A variants showing the same activity of wild type in inhibitingHMGB1-induced cell migration.

6.7 Results

The activity of polypeptide variants of the human HMGB1 high affinitybinding domain Box-A of SEQ ID NOs:2 to 116 was evaluated in comparisonto human HMGB1 Box-A wild-type of SEQ ID NO:1 as inhibition ofHMGB1-induced cell migration.

The chemotaxis assays results revealed (FIGS. 12.1 to 12.9) that for 26polypeptide variants the mutation according to the present inventioncould lead to a higher activity in cell migration inhibition incomparison with the activity of the wild-type human HMGB1 Box-A. Inparticular, a higher activity in cell migration inhibition was shown forthe polypeptide variants of SEQ ID NOs: 30-32, 35, 38, 40-41, 43, 48,51, 57, 63-64, 69, 70, 94, 95, 100, 103-104,106-107, 109-111 and 113.

Moreover, the chemotaxis assays results revealed (FIGS. 12.1 to 12.9)that 41 polypeptide variants showed no changes in their activity ininhibiting HMGB1-induced cell migration compared to the activity ofBox-A wild-type polypeptide. In particular, this is the case for thepolypeptide variants of SEQ ID NOs: 2, 7, 9, 11, 15, 18, 22, 26-28, 33,37, 39, 42, 44-47, 49, 52, 55, 59, 61, 62, 66-67, 71, 80-84, 86-87,97-99, 101-102, 112 and 114.

All these Box-A polypeptide variants which exhibit a similar or a higheractivity than the Box-A wild-type were tested for in vitro proteaseresistance, in order to choose the most resistant ones that are at leastas active as Box-A wild-type (see protease resistance test in Example7).

7. In Vitro Protease Resistance Testing

The purpose of the present study was to evaluate the in vitro proteaseresistance of to HMGB1 Box-A variants shown in Example 6 and to compareit to that of wild type HMGB1 Box-A of SEQ ID NO:1 in order to identifythe variants with improved protease resistance with respect to wild typepolypeptide.

7.1 Materials

HMGB1 His-tagged Box-A wild type and selected variants (Nautilusbiotech)

Trypsine (Sigma; cat. n. T8658; lot.n. 045K5113)

α-chymotrypsine (Sigma; cat. n. C6423; lot.n. 109H74858),

Endoproteinase Asp-N (Sigma; cat. n. P3303; lot.n. 046K1049)

Endoproteinase Glu-C(Sigma; cat. n. P6181; lot.n. 075K5100)

Complete, Mini EDTA-free protease inhibitor cocktail (Roche; cat. n.11836170 001)

Trizma base (Sigma; cat. n. T6066)

Acrilamide/bis solution 40% in water (Sigma; cat.n. 01709)

SDS (Sigma; cat. n. 71729)

Glycerol 99% (Sigma; cat. n. G9012)

Temed (Sigma; cat. n. 87689)

APS (Sigma; cat. n. A 3678)

Polypeptide SDS-PAGE Molecular Weight Standards (Bio-Rad; cat. n.161-0326)

Premixed 10× Tris/tricine/SDS Buffer (Bio-Rad; cat. n. 161-0744)

β-Mercaptoethanol (Sigma; cat. n. M7154)

Methanol (VWR; cat. n. 20864.320)

Acetic acid (VWR; cat. n. 20104.323)

Brilliant Blue R (Sigma; cat. n. B0149)

Bromophenol Blue (Sigma; cat. n. B0126)

Hydrochloric acid (Merck; cat. n. 1.00319.2511)

3× sample loading buffer for Tricine gels (composition: 150 mM Tris-HCl,pH 6.8; 12% SDS; 36% glycerol; 6% β-Mercaptoethanol; 0.04% ofbromophenol blue)

7.2. Protease Mixture Preparation

A mixture of proteases containing trypsine, α-chymotrypsine,endoproteinase Asp-N and endoproteinase Glu-C is used. Table 1 reportsspecificity of each of the proteases used in this study. TABLE 1protease specificity. Protease Specificity Tripsin C-term of K, R (notif P at C-term of cutting site; slower digestion if acidic residue oneither side of cutting site) α-chymotrypsin C-term of T, P, W, L(secondary hydrolysis: C-term of M, I, S, T, V, H, G, A) EndoproteinaseAsp-N N-term of D, C Endoproteinase Glu C-term of E, D (not if P is atC-term of cutting site)

Each lyophilized protease is dissolved according to manufacturerrecommendations to obtain a stock solution that is aliquoted and storedat −80° C.

100 μg of trypsin are dissolved in 100 μl of dH₂O to obtain a 1 μg/μlstock solution. 25 μg of α-chymotrypsine are dissolved in 50 μl of asolution 1 mM HCl, 2 mM CaCl₂ to obtain a 0.5 μg/μl stock solution. 2 μgof endoproteinase Asp-N are dissolved in 50 μl of dH₂O to obtain a 0.04μg/μl stock solution. 25 μg of endoproteinase Glu-C are dissolved in 50μl of dH₂O to obtain a 0.5 μg/μp stock solution.

Before performing the experiment one aliquot of each protease stocksolution is left to thaw on ice.

Trypsin and endoproteinase Glu-C stock aliquots are diluted in dH₂O toobtain a final working solution of 0.1 μg/μp. α-chymotrypsine stockaliquot is diluted in a solution 1 mM HCl, 2 mM CaCl₂ to obtain a final0.1 μg/μl working solution. Endoproteinase Asp-N aliquot is used withoutdilution.

Just before performing the experiment a mixture of proteases containing1% (in weight/weight of total Box-A contained in the sample) of eachprotease is freshly prepared and immediately added to HMGB1 Box-A to bedigested.

7.3. HMGB1 Box-A Wild Type and Variants Protease Digestion

18 μg total of each HMGB1 Box-A (wild type or variants) are digested ineach experiment.

HMGB1 Box-A to be tested is left to thaw on ice and the volumecorresponding to 18 μg is taken. The volume of this solution is thenbrought with dH₂O to a final volume of 90 μl in order to obtain the samefinal volume for each HMGB1 Box-A to be tested.

10 μl of this solution (corresponding to 2 μg of HMGB1 Box-A) are takenbefore adding the protease mixture. This sample corresponds to “time 0”not digested sample.

The remaining sample (16 μg of HMGB1 Box-A) is added with 8.8 μl(corresponding to 0.16 μg of each protease of the freshly preparedmixture; see 7.2) of protease mixture for digestion.

Protease digestion is performed at 25° C. and a volume corresponding to2 μg of HMGB1 Box-A (originally present in the mixture) is sampled atdefined time points. Digestion is stopped adding 4 μl of a solution ofcomplete Mini EDTA-free protease inhibitor cocktail (1 tablet dissolvedin 10 ml of dH₂O).

Timepoints for sampling are: 0, 5 minutes, 15 minutes, 30 minutes, 1hour, 1.5 hours, 2 hours and 4 hours.

Soon after protease inhibition, samples are added with the appropriateamount of sample loading buffer 3× and incubated at 95° C. for about 3minutes.

7.4. Tricine SDS-Page of Digested HMGB1 Box-A Wild Type and Variants

After protease digestion and samples preparation, timepoints samples ofeach HMGB1 Box-A are loaded on a Tricine SDS PAGE gel (see forreferences: Schägger and von Jagow, “Tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis for the separation ofproteins in the range from 1 to 100 kDa”, Anal. Biochem. 166, 368-379,1987).

5 μl of Polypeptide SDS-PAGE Molecular Weight Standards (Bio-Rad) areloaded for reference on each gel.

Each well of the gel is loaded with 10 μl of sample (volumecorresponding to 1 μg of HMGB1 Box-A before digestion).

Electrophoresis is performed at 30 V until the bromophenol blue hasentered the separating portion of the gel, then at 120 V (Mini Protean 3System; Bio-Rad) till the end of the run.

Gels are stained by soaking in a Coomassie Brilliant Blue R stainingsolution (0.1% w/v in 50% methanol, 10% acetic acid) for 1 hour anddestained overnight in destaining solution (30% methanol, 10% aceticacid).

Gel images are acquired with Gel Doc 2000 (Bio-Rad) imaging system.

7.5 Results

In the above reported assay conditions HGMB1 wild-type protein resistedapproximately 30 minutes to complete protease digestion. In FIG. 13 theband corresponding to the 84-amino acid full-length fragment of humanHMGB1 Box-A wild-type of SEQ ID NO:1 protein is visible until 30 minutesof protease digestion.

21 Box-A polypeptide variants tested showed an increased resistance toprotease (FIG. 15). In the reported assay conditions these variantsresist from 1 hour to 2 hours to protease digestion. The polypeptidevariants of SEQ ID NOs: 33, 35, 37, 38, 39, 42, 43, 44, 47, 48, 57, 62,69 and 104 showed a resistance of 1 hour to protease digestion. FIGS.14.14, 14.15, 14.16, 14.17, 14.18, 14.21, 14.22, 14.23, 14.26, 14.27,14.32, 14.35, 14.40 and 14.59 show a band corresponding to the notHis-tagged protein of 84 amino acids which is visible until 1 hour ofprotease digestion.

The polypeptide variants of SEQ ID NOs: 45, 49, 52, 55 and 67 showed aresistance of 1.5 hours to protease digestion. FIGS. 14.24, 14.28,14.30, 14.31 and 14.39 show a band corresponding to the not His-taggedprotein of 84 amino acids which is clearly visible 1 hour and a halfafter protease digestion. The polypeptide variants of SEQ ID NOs: 59 and64 even show a resistance of up to 2 hours to protease digestion. FIGS.14.33 and 14.37 show a band of the not His-tagged protein of 84 aminoacids which is clearly visible until 2 hours after protease digestion.

REFERENCES

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1. Polypeptide variant of the human and/or non human HMGB1 high affinitybinding domain Box-A (HMGB1 Box-A) or of a biologically active fragmentof HMGB1 Box-A, characterised in that the amino acid sequence of saidpolypeptide variant differs from the amino acid sequence of the wildtype HMGB1 Box-A by the mutation of one or more single amino acid. 2.Polypeptide variant of claim 1, wherein the polypeptide variant differsfrom the wild type HMGB1 Box-A sequence by the mutation of 1 to 10single amino acid, preferably by only one single amino acid. 3.Polypeptide variant of claim 1, wherein the mutation is a substitution,a deletion or an addition of single amino acids.
 4. Polypeptide variantof claim 3, wherein the substitution is obtained by differentgenetically encoded amino acid or by non-genetically encoded aminoacids.
 5. Polypeptide variant of claim 3, wherein the substitution is aconservative or a non-conservative substitution.
 6. Polypeptide variantof claim 1, wherein non-human HMGB1 Box-A is Anopheles gambia HMGB1Box-A.
 7. Polypeptide variant of claim 1, wherein the polypeptidevariant of the human HMGB1 Box-A is selected from the group consistingof the amino acid sequences as defined in any of SEQ ID NO:2 to
 116. 8.Polypeptide variant of claim 1, wherein the biologically activefragments of the human wild type HMGB1 Box-A is a fragment of at least77 or at least 54 amino acids respectively and comprises the amino acidsequences as defined in SEQ ID NO:117 or 223 respectively. 9.Polypeptide variant of claim 7, wherein the polypeptide variant of thebiologically active fragments of the human HMGB1 Box-A is selected fromthe group consisting of the amino acid sequences as defined in any ofSEQ ID NO:118 to 222 or 224 to
 300. 10. Polypeptide variant of claim 1,wherein the polypeptide variant of the Anopheles gambia HMGB1 Box-A isselected from the group consisting of the amino acid sequences asdefined in any of SEQ ID NO:302 to
 418. 11. Polypeptide variant of claim1, wherein the biologically active fragments of the Anopheles gambiawild type HMGB1 Box-A is a fragment of at least 77 or at least 54 aminoacids respectively and comprises the amino acid sequences as defined inSEQ ID NO:419 or 530 respectively.
 12. Polypeptide variant of claim 11,wherein the polypeptide variant of the biologically active fragments ofthe Anopheles gambia HMGB1 Box-A is selected from the group consistingof the amino acid sequences as defined in any of SEQ ID NO:420 to 529 or531 to
 612. 13. Polypeptide variant of claim 1, wherein said polypeptidevariant exhibits an increased resistance to the proteolytic activity ofproteases compared to the wild type HMGB1 Box-A or to the biologicallyactive fragment of the wild type HMGB1 Box-A.
 14. Polypeptide variant ofclaim 1, wherein the increase in resistance to proteolysis is in respectto at least one protease selected from the group comprisingchymotrypsin, trypsin, endoprotease, endopeptidase or a combinationthereof.
 15. Polypeptide variant of claim 1, wherein the increase inresistance to proteolysis is at least 10%, 20%, 30%, 40%, 50%, 70%, 80%,90%, 95% or more compared with the wild type HMGB1 Box-A. 16.Polypeptide variant of claim 1, wherein the polypeptide variant exhibitsa longer half life in body fluids compared to the wild type HMGB1 Box-Aor to the biologically active fragment of the wild type HMGB1 Box-A. 17.Polypeptide variant of claim 16, wherein the half life is at least 10minutes, 20 minutes, 30 minutes, 60 minutes or even longer compared tothe wild type HMGB1 Box-A.
 18. A nucleic acid molecule encoding apolypeptide variant as defined in claim
 1. 19. A vector comprising anucleic acid molecule of claim
 18. 20. A method for producing apolypeptide variant of claim 1, comprising: (i) introducing a nucleicacid molecule of claim 18 into a host; and (ii) culturing the cell,under conditions in which the encoded polypeptide variant is expressed.21. A method for producing a polypeptide variant of claim 1 usingchemical peptide synthesis.
 22. Polypeptide variant of claim 1 for theuse as active agent in a medicament.
 23. Use of a polypeptide variant ofclaim 1 for the manufacture of a medicament for the prevention ortreatment of HMGB1-associated pathologies or pathologies associated withHMGB1 homologous proteins.
 24. The use of claim 23, wherein theHMGB1-associated pathologies and the pathologies associated with HMGB1homologous proteins are pathological conditions mediated by activationof the inflammatory cytokine cascade.
 25. The use of claim 23, whereinthe pathological conditions are selected from the group consisting ofinflammatory disease, autoimmune disease, systemic inflammatory responsesyndrome, reperfusion injury after organ transplantation, cardiovascularaffections, obstetric and gynecologic disease, infectious (viral andbacterial) disease, allergic and atopic disease, solid and liquid tumorpathologies, transplant rejection diseases, congenital diseases,dermatological diseases, neurological diseases, cachexia, renaldiseases, iatrogenic intoxication conditions, metabolic and iodiopathicdiseases, and opthalmological diseases.
 26. The use of claim 23 incombination with a further agent capable of inhibiting an early mediatorof the inflammatory cytokine cascade.
 27. The use of claim 26, whereinthe further agent is an antagonist or inhibitor of a cytokine selectedfrom the group consisting of TNF, IL-1α, IL-1β, IL-R_(a), IL-6, IL-8,IL-10, IL-13, IL-18, IFN-_(γ), MIP-1α, MIF-1β, MIP-2, MIF and PAF. 28.The use of any of claims 26, wherein the further agent is an antibody toRAGE, a nucleic acid or nucleic acid analogue capable of inhibiting RAGEexpression, e.g. an antisense molecule, a ribozyme or a RNA interferencemolecule, or a small synthetic molecule antagonist of the HMGB1interaction with RAGE or soluble RAGE (sRAGE).
 29. The use of any ofclaims 26, wherein the further agent which is an inhibitor of theinteraction of a Toll-like receptor (TLR), in particular of TLR2, TLR4,TLR7, TLR8 or/and TLR9, with HMGB1, preferably a monoclonal orpolyclonal antibody, a nucleic acid or nucleic acid analogue capable ofinhibiting TLR expression, e.g. an antisense molecule, a ribozyme or aRNA interference molecule, or a synthetic molecule having a size of lessthan 1000 Dalton.
 30. The use of any of claims 26 wherein the furtheragent is the N-terminal lectin-like domain (D1) of native or mutatedthrombomodulin.
 31. The use of claim 26, wherein the further agent is asynthetic double-stranded nucleic acid or nucleic acid analogue moleculewith a bent shape structure, selected from bent or cruciform DNA, PNA orDNA/PNA chimeria or hybrid.
 32. The use of claim 26, wherein the furtheragent is K-252a or/and a salt or a derivative thereof or a polymerconjugate of K-252a or/and a derivative thereof.
 33. A pharmaceuticalcomposition comprising an effective amount of at least one polypeptidevariant of claim 1 as an active agent and optionally a pharmaceuticallyacceptable carrier.
 34. The composition of claim 33 wherein the at leastone polypeptide variant is in combination with at least one furtheragent.
 35. The composition of claim 33 for diagnostic applications. 36.The composition of claim 33 for therapeutic applications.
 37. A methodof treating a condition in a patient, characterized by HMGB1-activationof an inflammatory cytokine cascade, comprising administering to thepatient an effective amount of at least one of the polypeptide variantsof claim 1, capable of antagonize and/or inhibit the pathologicalactivity induced by HMGB1.
 38. The use of at least one polypeptidevariant of claim 1, wherein said molecules are reversibly immobilised onthe surface of medical devices.
 39. The use of claim 38, wherein saidmedical devices are surgical instruments, implants, catheters or stents.40. Medical device reversibly coated with at least one polypeptidevariant according to claim
 1. 41. Medical device of claim 40, whereinthe medical device is selected from surgical instruments, implants,catheters or stents.